Biologically active rupununines

ABSTRACT

A method of therapy wherein there is administered to a person in need of such therapy a pharmacologically effective amount of a rupununine having the formula: ##STR1##

This application is a continuation-in-part of application of Ser. No. 08/189,781, filed Feb. 1, 1994, U.S. Pat. No. 5,569,456.

The invention relates to alkaloids, and especially to bisbenzylisoquinoline alkaloids, and derivatives thereof.

It has been known for some time that Amerindian peoples of the Rupununi area of Guyana, South America chew the nuts of the greenheart tree (Ocotea rodiaei) as a crude form of contraception. Also, infusions of the bark of the greenheart tree have been used as a febrifuge and as an anti-periodic in fevers. Some bisbenzylisoquinoline alkaloids from other plants are known to have similar uses, and it was considered possible that the activity of the greenheart tree was attributable to a bisbenzylisoquinoline alkaloid. Although bisbenzylisoquinoline alkaloids have been extracted from the greenheart tree, no biological activity had previously been reported for such alkaloids.

We have now isolated an active bisbenzylisoquinoline alkaloid which we have named rupununine C₃₇ H₄₀ O₆ N₂ which has now been characterised, as in Formula 1. The isolated compound had OR₁ and OR₂ one as a hydroxy group the other as a methoxy group, not distinguished in the mass spectrograph.

The structure of the rupununines of the invention is as follows; ##STR2## wherein R═--H or --CH₃, R₁ and R₂ are optional derivatisations the same or different and are thus for example --H, alkyl, especially --CH₃, acyl or glycosidyl. The nitrogens may of course be quaternised and in such form are regarded as within the above formulae.

Rupununine is a bisbenzylisoquinoline alkaloid. Related alkaloids not forming part of the invention, include tertiary alkaloids such as curine and isochondrodendrin. Many tertiary alkaloids of this series show biological properties. For example, thalicarpine (isolated from Thalictrum dasycarpum) is a hypotensive and cytotoxic agent. Cissampereine (isolated from Cissampelos pareira) exhibits cytotoxic action and tetrandrine (isolated from Cyclea peltata) is known to be a tumour-inhibitory alkaloid.

Although the greenheart tree (Octotea rodiaei) has been reported to be a source of d-curine, no biological activity has been reported of bisbenzylisoquinoline alkaloids from this plant. However it is known that amongst the Wapishana tribe of the Rupununi area of Guyana, the seeds (or fruits) of this tree have been used as a form of oral contraceptive. The fact that the alkaloids rodiasine, sepeerine, ocotine, ocotosine, demerarine, dirosine, norrodiasine and 2(+)- nortetrandrine have been isolated from the bark and seeds of this tree, brought speculation as to the possible biological properties existing in the plant.

The greenheart tree, Octotea rodiaei, is a large tree with a gently tapering erect stem, which reaches a height of approximately 60 to 90 feet and is branched only at the summit. The tree has a circumference of between 9 and 12 feet, and is recognised by its dense, glossy foliage and comparatively white flowers. The bark of the tree is smooth and whitish grey. The tree only grows in Guyana (formerly British Guiana), and specimens of the tree and its nuts may be found in the Herbarium at the Royal Botanic Gardens, Kew.

Ground defatted greenheart nuts may be extracted with for example, methanol, or isopropanol or with distilled water in an aspirator. The extract may then be reduced to a syrup for example, using a rotary evaporator, and the syrup was diluted and further extracted. Column chromatography may follow and the extract from the column further separated for example in a cascade partition chromatography solvent system. Counter-current distribution studies and Craig Distributions may then be carried out.

Crystals of rupununine have been isolated after preparative TLC and repeated recrystallisation in e.g. acetone affords white needles which melt at between 214 and 217° C. The ultra violet spectrum of rupununine exhibits the characteristic absorption band at 283 nm corresponding to its aromatic chromophore (approximately 282 nm for most BBI's) with a second intense band at 206 nm. The molecular weight of rupununine where R is hydrogen, is approximately 608, and where R is methyl is approximately 622. The optical rotation [α]_(D) (CHCl₃), of rupununine is +257±10.

Infra-red spectroscopy reveals the presence of a doublet between 1500 to 1580 cms⁻¹ confirming that the alkaloid has a 1,2,3,4-tetrahydroisoquinoline base. The presence of a free hydroxyl is suggested by a weak singlet at about 3550 cms⁻¹.

The applicants conclude that this rupuninine is responsible for certain of the effects traditionally seen following native use of extracts of the greenheart tree.

The present invention embraces the use of rupununine in the treatment of disease. For example, rupununine may be useful in the treatment of malaria, especially as a febrifuge and anti-periodic and in topical application for skin lesions. Rupununine is also thought to be useful as a contraceptive or anti-fertility active, possibly with a role in the control of oestrus. This anti-fertility activity is probably highly selective for humans. Other applications of rupununine include a use as an inhibitor of soft tumour development, and possibly in the control of viruses such as AIDS and as an antimitotic through an antimetastic role or generally through a role in the cell proliferative process. Rupununine affects the neuropathways, and provides benefits in conditions that affect neurofunction, possibly as a treatment for intractable CNS disorders especially at a neurochemical level with GABA mediation. Its amphibatic properties suggest membrane action and an ability to pass cell barriers especially the blood brain barrier.

The present invention thus relates in general to the beneficial effects of rupununine in neurofunction.

Further uses envisaged are as a pesticide or preservative; especially for timber, nets or other materials in a marine environment or for example as an anti-fouling agent for boats and other structures, against barnacles, etc. The compound is applied to the pest or to a substrate affected by it or to be protected from it, or to a substrate to be preserved generally.

Derivatives of rupununine are envisaged, where the molecule of formula 1 is derived at R₁ and R₂. Suitable derivatives include those imparting polarity or lipophilicity to the molecule, for example to aid transfer of the molecule across cell membranes. A wide range of substituents is known, and selection may be made from those acceptable in themselves and also in the sense of allowing the desirable properties of the free compound still to be shown.

Derivatives of rupununine according to the invention may include glycosides, alkyl derivatives, especially methyl, acyl derivatives, derivatives of essential fatty acids, such as gamma-linolenic acid and dihomo-gamma-linolenic acid and the other n-6 and n-3 essential fatty acids whether in the form of the acids or as the corresponding fatty alcohols.

The invention is further illustrated by the following example.

EXAMPLE Methanol Extraction

Ground nuts (2 kg) of O. rodiaei, defatted using petroleum ether 60/80, were loosely packed into an aspirator (ca 5 liters), which was fitted with a tap plugged with cotton wool. This filter device retained the fine powdered particles during extraction with solvent. The vessel was then filled to the top with distilled methanol (2.51), and left to soak overnight (16 hours) in a dark place. The methanol extract was then allowed to run off, effecting a continuous extraction until a total volume of 12 liters of extract was obtained. This was reduced to a dark syrup on the rotary evaporator under reduced pressure using a water pump, giving a syrupy methanol extract (200 g).

Distilled water (800 ml) was added to the concentrated syrup (180 g) to give a dark brown solution (pH=5). Ammonia (0.880,2 ml.) was added to adjust the pH of the solution to a pH of about 8. The solution was then divided into two portions of approximately 450 ml. each.

The two portions were then shaken exhaustively with distilled chloroform (450 ml portions) in stoppered glass jars on a shaker for about 30 minutes.

The two layers obtained were separated with a separating funnel to give an orange-coloured chloroform layer, and a thick brown aqueous layer. The partitioning with chloroform was repeated until the total volume of chloroform extract obtained was 3.51.

The chloroform layer was dried over anhydrous sodium sulphate (40 g), filtered and evaporated to dryness on the rotary evaporator to give a brown syrup: chloroform extract (50.8 g, Fraction B), which was kept under nitrogen in the refrigerator until required. TLC using methanol: chloroform (1:10 v/v) indicated the presence of at least 5 alkaloidal components (detected by Dragendorff's reagent). The aqueous layer on drying gave a brown solid (95 g), and showed the presence of three alkaloidal substances by TLC using the same system as before.

Column chromatography of the chloroform extract (Fraction B)

A column (diameter 4 cm×length 77 cm) was packed using neutral alumina (60 g; Merck) as a slurry in chloroform (distilled). This was then equilibrated using the eluting solvent, distilled bench chloroform. The chloroform extract, Fraction B (50 g) was dissolved in the minimum amount of chloroform (50 ml), and loaded onto the column. The elution was carried out at 10 ml/min, and monitored by TLC.

Aliquots of 500 mls., of the eluted solvent were collected to give 3 main fractions which were quite well separated.

                  TABLE 1                                                          ______________________________________                                                      Aliquot         Weight                                                         No      Volume  (Dried)                                           ______________________________________                                         FRACTION B.1   1,2,3     1500 ml 10 g                                          FRACTION B.2   4,5,6     1500 ml 9 g                                           FRACTION B.3   7,8,9,10  2000 ml 4 g                                           FRACTION B.4   11,12,13  1500 ml 7 g                                           (Methanol Fraction)                                                            ______________________________________                                    

TLC showed that fraction B.1 had the most alkaloidal content compared to the later fractions.

The solvent system was changed to pure methanol when very little alkaloidal substance seemed to be eluting through the column (monitored by TLC). The methanol fraction (Fraction B.4, 7 g) had similar Rf to fraction B.3. The dried fractions were pale yellow powders; and they were stored under nitrogen in the cold.

Partition Chromatography: Cascade System

A solvent system consisting of 60/80 petroleum ether:distilled water:distilled butanol (3:4:3 v/v) was prepared. This mixture was shaken and left to equilibrate at room temperature. The two phases separated out fairly easily within a minute. The upper phase (petrol layer) was distributed into five separating funnels so that each funnel had 100 ml., of solvent. The lower phase was treated likewise into another five separating funnels.

The chloroform extract (fraction B, 5.6 g) was dissolved in upper phase (100 ml.) by shaking vigorously for 25 minutes on a shaker. The solution was then introduced into a separating funnel containing lower phase (100 ml.). The mixture was shaken well and left to equilibrate for five minutes. The two phases separated out during this time into an orange coloured upper phase and a less coloured lower phase. The upper and lower phases were then partitioned successively into the appropriate separating funnels prepared previously.

The procedure gave a separation of dark orange fraction in the upper phase and a paler fraction in the lower phase (butanol layer). TLC with methanol:chloroform (1:10 v/v) indicated that the upper phase (petrol layer) was richer in alkaloids (4 or 5 positive Dragendorff's spots), than the butanol layer (3 spots). After removal of solvent, the petrol layer (3.6 g) and butanol layer (1.5 g) gave dark brown solids and were stored under nitrogen in the fridge.

The experiment formed the basis of the Counter-current distribution studies and Craig distribution.

Counter-Currrent Distribution Studies: Solvent System

A solvent system consisting of petroleum ether 60/80 (distilled), distilled water and distilled butanol (3:4:3 v/v) was mixed in an aspirator (capacity 15.1) by stirring using an induction electric motor with a metal stirrer for four hours. The aspirator was kept covered to minimise evaporation of solvent, and later left to equilibrate in the Steady State Counter Current Distribution Machine (SSDM) room until used. The two phases were separated using a separating funnel (5 l). The solvents (15 l) were loaded into the two appropriate reservoirs (upper and lower phases) of the SSDM.

Solubility of Fraction B

The sample used was Fraction B.1 (Chloroform extract). The solubility in upper phase solvent was found to be 0.5 g/ml. For the study it was possible to dissolve approximately 9 g in 25 ml., of upper phase solvent. This was done by vigorously shaking the sample (10 g) in the upper phase solvent (30 ml) for an hour, on a shaker, to dissolve the maximum amount of sample in the solvent.

A Craig Distribution was then attempted in order to determine the partition coefficients, K, of the various constituents of the chloroform extract, Fraction B.1. The extract (9 g) dissolved in 25 ml. of the upper phase was introduced into tube 0, and a Craig Distribution was set up using a 1:1 lower phase to upper phase program for 98 transfers. The SSDM was programmed to have a three minutes agitation time and 5 minutes settling time.

After 98 transfers were completed the train was analysed by sampling a series of tubes (every 3rd tube), and their absorbances were determined at λ maximum 283 nm after appropriate dilution.

A TLC analysis was also attempted in the same manner (on the upper bank only: tubes no. +1 to +49). This was done after reducing the samples to a small volume (0.5 ml). An activated silica gel (60 G P254) plate of size 20 cm×20 cm was developed as in system TLC.1. The alkaloidal constituents were visualised using Dragendorff's reagent.

A distribution of the main fraction (Fraction C.1, 5.1 g) from tubes +46 to +41 was obtained which gave a Kp value of 15.33. TLC suggested 2 alkaloids of R.f.s 0.36 and 0.23. The contents of tubes +46 to +41 (Fraction C.1) were removed and the solvent removed. The rest of the train was also removed including another minor component of Kp 1.28 and solvent evaporated off.

                  TABLE 2                                                          ______________________________________                                                                            PARTITION                                                       PEAK           COEFFICIENTS                                TUBE NO.  FRACTION  TUBE    WEIGHT Kp                                          ______________________________________                                         +46 to +41                                                                               C.1       +43     5.09 g 15.33                                       +49 to +47                                                                               C.2       +46     0.73 g 31.66                                       +40 to +35                                                                               C.3       +40     0.63 g 9.88                                        +34 to +26                                                                               C.4       +26     0.04 g 3.26                                        +25 to +8 C.5       +19     0.03 g 2.26                                        +6 to 0   C.6       +6      0.08 g 1.28                                        -49 to -1 C.7       -32     0.03 g 0.21                                        (LOWER BANK)                                                                   ______________________________________                                    

The study showed that almost 80% of the sample moved to the right of the cell train with the upper phase (K value greater than 1). Hence the portions of interest in the SSDM train were tubes +46 to +41 (Fraction C.1).

Isolation of Rupununine--Preparative TLC:Apparatus

Long plates (1 m×20 cm) were coated with silica gel (60 G P254 Merck) and activated in a special one-metre oven overnight. The oven was also used for storing the plates after setting. The developing tank was a stainless steel lidded tank (1.10 m×17 cm×24 cm), and was lined with chromatography paper to ensure uniform vapour saturation on developing. A specially designed applicator or spreader was used to operate on a Shandon TLC spreader track when loading the sample. This device allowed the application of the dissolved sample as a streak on the coated plate, via an Agla syringe, on running the applicator down the length of the track.

The plates were developed with methanol:chloroform:diethylamine (14:85:1 v/v). The sample (Fraction C.1, 640 mg) was dissolved in distilled chloroform (4 ml.), and applied onto the plates (using the spreader) as a streak: A loading of approximately 80 mg per plate gave a yield of about 40 mg., rupununine, and recover of about 78% of starting material. The developed plates were viewed under ultra violet light (λ 254 nm, 350 nm), and two distinct bands could be observed. These bands were both positive to Dragendorff's reagent.

The bands (Band I, Band II) (Rfs. 0.3, 0.4), were marked with a scribe, and scraped off the plate. The silica scrapings were then extracted with warm methanol leaching through the silica in a small column. The column was plugged tightly with cotton wool to trap the fine particles of silica. The solvent was removed on the rotary evaporator giving a pale yellow solid, rupununine (326 mg.) and O.R.II (176 mg.) Rupununine constituted about 0.16% of the original ground nuts of O. rodiei in weight.

Crystallisation

Rupununine (100 mg.) was dissolved in the minimum volume of distilled acetone (2 ml.). The solution was warmed gently, filtered and then left in the refrigerator overnight. This produced white needles which were recrystallised in acetone giving crystalline rupununine (45 mg.). Further crystalline samples were also obtained by the same treatment with acetone, of Fraction C.1 and C.2. The crystals were dried in a drying pistol under nitrogen, over phosphorus pentoxide at 60° C. overnight. The crystalline white solid was fairly soluble in methanol, ethanol and chloroform, but only partially soluble in ether. The crystals melted between 214-217° C., and were fairly stable, if kept under nitrogen at room temperature, even for prolonged periods.

Repeated recrystallisation on the crystals produced identical needle-like crystals with constant melting point (214-217° C.). Attempted recrystallisation in various other solvents, apart from acetone (such as ethanol, methanol, chloroform and ether) was unsuccessful.

Derivatisation is by procedures standard in themselves, for example:

METHYLATION OF RUPUNUNINE USING DIAZOMETHANE Experimental

Dried, crystalline rupununine (200 mg) was dissolved in methanol (2 ml) and an ethereal solution was added in small portions until there was an excess of the reagent and left to stand at room temperature for five days. The excess diazomethane was removed by distillation. A pale yellow solid was obtained (208mg) which melted between 127-129° C. The reaction product was a mixture from which the methylated rupununine was isolated using preparative thin layer chromatography using silica gel (Merck 60 G p254) developed in methanol:chloroform (1:10 v/v).

Physical Data

O-Methyl rupununine melted between 140-145° C.

Chemical shifts (ζ) of the O-Methyl and N-Methyl protons in O-Methyl rupununine.

    ______________________________________                                         O--Me                       N--Me                                              13     6          6'     7        2'   2                                       ______________________________________                                         3.97   3.78       3.41   3.23     2.66 --                                      3.95   3.77       3.42   3.21     2.66 --                                      ______________________________________                                    

The Mass spectra yielded m/z 622 consistent with the expected methylation product of a 608 molecular weight compound with one free hydroxyl.

ACTIVITY OF RUPUNUNINE

The use of rupununine in the treatment of disease is illustrated by the following cytotoxicity studies.

Preliminary cytotoxicity studies were carried out on Chinese hamster lung tumour cells previously cultured in a growth medium. Rupununine was converted into the hydrochloride by purging HCl gas into a solution of rupununine (50 mg) in dry chloroform. The HCl gas was generated by the addition of concentrated hydrochloric acid to concentrated sulphuric acid (using a dropping funnel). The hydrochloride was then dried by removal of solvent on the rotary evaporator, and then dissolved in normal saline with appropriate dilution. Eight different concentrations of rupununine hydrochloride were prepared (7.7×10⁻³ M to 1.5×10⁻⁶ M).

The growth medium in the bottle containing the Chinese hamster lung tumour cells were removed, and the cells treated with trypsin to stop protein synthesis. The bottle was then left to incubate at 37.5° C. for 10 minutes to give trypsin time to act. The trypsin was removed and an equal volume of medium (GIBCO) added, and the mixture pipetted vigorously. A cell count was attempted on a sample of the cell population in the bottle. This gave a cell count of 1.2×10⁵ cells/ml.

The eight concentrations of rupununine hydrochloride were pipetted into 2 series of 8 tubes, leaving another 2 tubes as control tubes. The tubes were labelled (series 1: Tubes 1-8 and C; Series 2; Tubes 1a-8a and Ca). Water (1 ml) was added to all tubes, and then equal volumes of cells (6 ml) and medium (6 ml) were pipetted into all tubes and controls. The bottles were then incubated for 10 days at 37.5° C.

After 10 days, the cells were stained with crystal violet, after the removal of medium etc. This is because a cell count was not possible because the cell population was too high, and the cells congregated into clones instead. The staining intensity in the different tubes indicated the level of cell death. This revealed a dose-related effect. At concentrations higher than 1.5×10⁻⁴ M, complete cell death occurred, and no staining was observed. Below this concentration, the intensity (cell population) was decreased as concentration of rupununine hydrochloride was increased. This indicated that the cytotoxic effect was increased with an increase in concentration. In control tubes, very little cell death seemed to have occurred. The results are shown in the table below.

                  TABLE                                                            ______________________________________                                         CYTOTOXIC EFFECTS OF RUPUNUNINE (O.R. 1)                                              Concentration of                                                        Tube Nos.                                                                             O.R.1. Hydrochloride                                                                         Stain Intensity*                                                                            Comments                                     ______________________________________                                         C; Ca  0             XXXX       |                                                                         CYTOTOXIC                                  1; 1a  1.5 × 10.sup.-6 M                                                                      XXXXX      |                                                                         EFFECT                                     2; 2a  7.7 × 10.sup.-6 M                                                                      XXXX       |                                                                         INCREASE                                   3; 3a  1.5 × 10.sup.-5 M                                                                      XXX        |                                     4; 4a  7.7 × 10.sup.-5 M                                                                      XX          |                                    5; 5a  1.5 × 10.sup.-4 M                                                                      X            ↓                                     6; 6a  7.7 × 10.sup.-4 M                                                                      0              COMPLETE                                   7; 7a  1.5 × 10.sup.-3 M                                                                      0               CELL                                      8; 8a  7.7 × 10.sup.-3 M                                                                      0              DEATH                                      ______________________________________                                          *Stain intensity indicated by number of X's.                             

From the above results, it is suggested that this alkaloid rupununine has cytotoxic activity, which appears to be dose-related within the concentrations used.

ADMINISTRATION EXAMPLES

The compounds may be administered orally, topically, parenterally (subcutaneously, intramuscularly, intravenously), enterally, rectally, vaginally or by any other appropriate route. They may be made up into tablets, hard or soft gel capsules, pastilles, emulsions, enteral or parenteral formulae, foams, ointments, creams, lotions, suppositories, pessaries or any other appropriate form known to those skilled in the art. They may be made up into pharmaceutical dosage forms, or into foods which have a specific medical or health-related purpose.

It will be understood that the absolute quantity of active materials present in any dosage unit should not exceed that appropriate to the rate and manner of administration to be employed but on the other hand should also desirably be adequate to allow the desired rate of administration to be achieved by a small number of doses. The rate of administration will moreover depend on the precise pharmacological action desired. For example, the dosage amounts will be dependent on whether the use is for targeting the whole body or specific tissue.

The dosage units may suitably be prepared so as to deliver from 1 microg. to 10 g, preferably from 500 microg to 1 g and very preferably from 1 mg to 100 mg of the rupununine or rupununine derivative per kg body weight.

The dosage units may contain concentrations of 0.1 microg to 100 mg/ml, preferably 1 microg to 10 mg/ml and very preferably 10 microg to 1 mg of the rupununine or rupununine derivative.

When prepared for topical administration or in enteral or parenteral formulations or food they may be made in the formulae containing from 0.01% to 60% by weight of the final formulation, preferably from 0.1% to 30% by weight, and very preferably from 1% to 10% by weight.

The preparations may be used in any disease condition likely to respond to the rupununine or rupununine derivative. 

What is claimed is:
 1. A method of treating lung tumors sensitive to rupununine wherein there is administered to a person in need of such therapy a pharmacologically effective amount of a rupununine having the formula: ##STR3## wherein R═--H or --CH₃ and wherein one or both of the hydroxy groups may carry a substituent, the same or different, selected from the group consisting of alkyl, acyl and glycosidyl groups.
 2. A method of therapy according to claim 1 wherein the rupununine is in the form of a derivative in which one or both of the hydroxy groups carries a substituent, the same or different, selected from the group consisting of alkyl, acyl and glycosidyl groups.
 3. A method of therapy according to claim 2, wherein said alkyl substituent is methyl. 